1. R O S H N I . P
M S C B I O C H E M I S T R Y
SiRNA : structure and function
2. What is RNA?
Ribonucleic acid
Ribonucleotides (Ribose, base, & phosphate)
Types
Coding: messenger RNA (mRNA)
Non-coding:
Ribosomal RNA (rRNA)
Transfer RNA (tRNA)
Small nuclear RNA (snRNA)
Small nucleolar RNA (snoRNA)
Interference RNA (RNAi)
Short interfering RNA (siRNA)
Micro RNA (miRNA)
3. siRNA : (small-interfering RNAs)
Function of the species is regulation of gene expression
siRNA originates with dsRNA
Approximately 21-28 nucleotides in length.
siRNA is most commonly a response to foreign RNA
(usually viral) and is often 100% complementary to the
target
4. siRNAs have a defined structure
19 nt duplex
2 nt 3’
overhangs
5. siRNAs found in nature are derived from the
cytoplasmic processing of long dsRNA by the RNase-IIItype
enzyme termed Dicer.Dicer cleaves long dsRNA into 21- to 28-
nucleotide siRNA duplexes that contain 2-nucleotide 3′
overhangs with 5′ phosphate and 3′ hydroxyl termini.
Components of the RNAi machinery specifically recognize the
siRNA duplex and incorporate a single siRNA strand into a
protein complex termed the RNA-induced silencing complex
(RISC). RISC cleaves mRNAs containing perfectly
complementary sequences, 10 nucleotides from the 5′ end of
the incorporated siRNA strand.
13. •RNAi is mediated by small interfering RNAs (SiRNAs) that are
generated from long dsRNA.
•Long dsRNAs are cleaved by a ribonucleaseIII (RNaseIII) type
protein Dicer.
•Dicer homologues can be found in S.pombe, C.Elegans,
Drosophila, plants, and mammals, suggesting that small RNA –
mediated regulation is evolutionarily ancient and may have
critical biological roles.
14. DICER
Dicer (class 3 RNAse III) cleaves long dsRNA
into siRNA 21-25nt dsRNA
Symmetric 2nt 3’ overhangs, 5’ phosphate groups
16. Dicer cuts dsRNA into short RNAs
Vanhecke, D.; Janitz, M. Drug Discov. Today, 2005, 10, 205-225.
Dicer, the RNase III enzyme that is evolutionarily conserved and
contains helicase and PAZ domains, as well as two dsRNA-binding
domains.
17.
18.
19. How do Dicer proteins work in
dsRNA processing?
The PAZ (Piwi Argonaute Zwille) and
RNase III domains play central roles in
excising siRNAs preferentially from ends of
dsRNA molecules.
20. PAZ domains are shared with Argonaute proteins and are
specialized to bind RNA ends, especially duplex ends with short
(2 nt) 30 overhangs. An end engages the Dicer PAZ domain, and
the substrate dsRNA then extends approximately two helical
turns along the surface of the protein before it reaches a single
processing center.The center resides in a cleft of an
intramolecular dimer involving the RNase III domains. Each of
the two RNase III active sites cleaves one of the two strands,
leading to staggered duplex scission to generate new ends with 2
nt 30 overhangs. The reaction leaves a 50 monophosphate on the
product ends, consistent with a requirement for this group during
later stages of silencing.
22. processing the dsRNA into 21-23 nt fragments
34
27
21
20
16
short-interfering RNA
Q uic kTime ™ a n d a
G IF d ec o mp re s so r
a re n ee d ed to s ee this p ic tu re .
Tuschl, 2001
23. RNAi silencing complex
• may be associated with translating ribosomes
• active RNAse enzyme not yet identified
• may participate in endogenous pathways that
silence genes via translational repression
24. RNA Induced Silencing Complex
(RISC)
RNAi effector complex
Preferentially incorporates one strand of
unwound RNA .
Antisense
How does it know which is which?
The strand with less 5’ stability usually incorporated
into RISC.
28. Basic research
Determining protein function
Easier than a knockout and may be used for partial
knockdowns
Clinical research
Cancer, hypercholesterolemia, infections, developmental
defects
29. siRNA for treatment of AMD
Age-related macular degeneration (AMD) is an eye disease that destroys
central vision by damaging the macula, the central region of the retina. AMD
affects millions of people worldwide. The main symptoms of AMD is dim or
fuzzy central vision. Objects may appear distorted or smaller then they really
are, and straight lines may appear wavy or curved. Patients may develop a
blank or blind spot in their central field of vision. There are no effective
therapies.
30. siRNA for treatment of AMD
AMD is often associate and promoted by
neovascularization - new blood vessel growth.
Macular neovascularization is stimulated by
interaction of vascular endothelial growth factor
(VEGF) with vascular endothelial growth factor
receptor VEGFR-1. Inhibiting production of
VEGFR-1 should stop neovascularization and
prevent development of AMD.
Inhibition of blood
vessel
growth in corneas
of mice
after a single
intravitreal
injection
31. siRNA targeting ApoB
ApoB is a liver enzyme essential for the assembly and secretion
of low-density lipoprotein (LDL), which are required for
metabolism of cholesterol. High levels of ApoB and LDL
increase risk of coronary artery disease. Alnylam
Pharmaceuticals developed siRNA to target ApoB. They first
used chloresterol-conjugated siRNA to demonstrate its effect on
ApoB production in mice.
36. SiRNA have become not an exciting new tool in nolecular
biology but also the next frontier in molecular medicine.
Significant hurdles remain, most notably guaranteeing
specificity and finding safe and efficacious delivery system.
Work is ongoing to solve these problem, but the therapeutic
promise of SiRNA remains great.
CONCLUSION